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. 2004 Jan;15(1):24-36.
doi: 10.1091/mbc.e03-05-0297. Epub 2003 Oct 3.

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase

Simon A Rudge  1 , Deborah M Anderson, Scott D Emr
Affiliations

Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase

Simon A Rudge et al. Mol Biol Cell. 2004 Jan.

Abstract

In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1. Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control. Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole. More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4. We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro. Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole. Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole. However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants. Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex. Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.

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Figures

Figure 1.
Figure 1.
Fig4 is a magnesium-activated PtdIns(3,5)P2-specific phosphoinositide phosphatase. (A) Recombinant His-tagged full-length Fig4 was assayed in the absence and presence of 1 mM magnesium chloride using fluorescent PtdIns(3)P and PtdIns(3,5)P2 as described in MATERIALS AND METHODS. Reaction products were analyzed by TLC, and the positions of the fluorescent phosphoinositides were visualized with ultraviolet light. (B) Recombinant His-tagged fig4-1 and Fig4 were assayed for phosphatase activity in the presence of 1 mM magnesium using fluorescent PtdIns(3,5)P2 as substrate. (C) Quantitation of His-tagged Fig4 phosphatase activity against fluorescent derivatives of all four known phosphoinositides synthesized in S. cerevisiae.
Figure 2.
Figure 2.
Fig4 localizes to the limiting membrane of the vacuole. Wild-type cells expressing (A) Fig4-GFP or (B) fig4Δ761–879-GFP were labeled with the fluorescent dye FM4-64 for 10 min at 26°C. The dye was washed away and the cells were chased for 60 min at 26°C. The cells were concentrated and chilled on ice, and the localization of FM4-64 and Fig4-GFP was compared by fluorescent microscopy.
Figure 3.
Figure 3.
Vac14 recruits Fig4 phosphatase to the limiting membrane of the vacuole. The vacuoles of (A) vac14Δ and (B) vac7Δ mutants expressing Fig4-GFP were visualized by FM4-64 staining as described in MATERIALS AND METHODS. The localization of FM4-64 and Fig4-GFP were compared by fluorescent microscopy. (C) The total cellular protein content of wild-type, vac7Δ, and vac14Δ mutants were precipitated with TCA and resolved by SDS-PAGE. Fig4-GFP was detected by Western blotting with anti-GFP antibody. Western blotting with anti-Vam3 served as a loading control.
Figure 4.
Figure 4.
Vac14 localizes to the limiting membrane of the vacuole. (A) Vac14-GFP–expressing wild-type cells were labeled with the fluorescent dye FM4-64 to visualize the vacuole membranes. The localization of FM4-64 and Vac14-GFP were compared by fluorescent microscopy. (B) The total cellular protein content of wild-type and wild-type expressing Vac14-GFP were TCA-precipitated and resolved by SDS-PAGE. Vac14-GFP was detected by Western blotting using anti-GFP antibody. (C) Quantitation of PtdIns(3,5)P2 in wild-type, vac14Δ, and VAC14-GFP strains. 3H-labeled phosphoinositides were isolated, resolved and measured as described in MATERIALS AND METHODS. The levels of PtdIns(3,5)P2 are expressed as a percentage of the total 3H-labeled phosphoinositides analyzed by HPLC.
Figure 5.
Figure 5.
Vac14 and Fig4 do not localize to the E compartment of vps4Δ mutants. The vacuoles and the E compartments of vps4Δ mutants expressing either Vac14-GFP (A) or Fig4-GFP (B) were visualized by FM4-64 staining. The localization of the GFP fusion proteins relative to the FM4-64–positive E compartments (highlighted by white arrow heads) were compared by fluorescence microscopy.
Figure 6.
Figure 6.
Vac14 localizes to the vacuole more efficiently when associated with Fig4. Vac14-GFP localization was determined in FM4-64–labeled fig4Δ (A) or vac7Δ (B) mutants by fluorescence microscopy.
Figure 7.
Figure 7.
Fab1 kinase is required for vacuole localization of Vac14 and Fig4. (A) Vac14-GFP and (B) Fig4-GFP localization were determined in FM4-64–labeled fab1Δ mutants by fluorescence microscopy.
Figure 8.
Figure 8.
Vac14 interacts with Fig4. Coimmunoprecipitation of Vac14-GFP and Fig4 from P13 and P100 fractions. Lysates from wild-type cells expressing Vac14-GFP were fractionated into P13, S100, and P100 fractions, and Vac14-GFP immunoprecipitated from detergent-solubilized fractions, as described in MATERIALS AND METHODS. Vac14-GFP was resolved by SDS-PAGE and visualized by Western blotting using anti-GFP antibody. Fig4 was detected by Western blotting with anti–Fig4 antisera.
Figure 9.
Figure 9.
Mislocalized Fig4 phosphatase does not contribute to vac14Δ phenotypes. Vacuoles of wild-type, fig4Δ, vac14Δ, and vac14Δ fig4Δ strains were visualized by FM4-64 staining and fluorescence microscopy.
Figure 10.
Figure 10.
Vac14 is required for fig4Δ suppression of vac7Δ. The vacuole morphologies of wild-type, vac7Δ, vac7Δ fig4Δ, and vac7Δ fig4Δ vac14Δ strains were visualized by fluorescent staining with FM4-64 and fluorescence microscopy.
Figure 11.
Figure 11.
Model. Vac14 regulates PtdIns(3,5)P2 phosphatase activity at the vacuole membrane by both positively regulating Fab1 kinase and controlling Fig4 phosphatase localization.
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